1. Field of the Invention
The present invention relates to a sample inspection apparatus, sample inspection method, and sample inspection system capable of inspecting a sample by irradiating the sample held on a film with a primary beam, such as a charged-particle beam. Especially, the present invention relates to techniques permitting one to observe reactions of a sample consisting of biological cells when a stimulus is given to the cells and to inspect the sample.
2. Description of Related Art
In life science and pharmaceutical applications, it is important that stimuli (such as electricity, chemical substances, and medicines) are given to biological cells and that resulting reactions are observed. In the past, optical microscopes have been used for such observations. Often, important parts to be observed are microscopic regions of less than 0.1 μm, which cannot be observed with optical microscopes.
For example, diseases arising from inability to exchange intercellular messengers (signaling molecules) among biological cells normally include hypertension, diabetes insipidus, arrhythmia, muscular disorders, diabetes, and depression. Exchange of these substances among cells is performed by membrane protein molecules, such as receptors and ion channels having sizes of about 10 nm and existing in cell membranes. Because it is difficult to observe such membrane protein molecules with optical microscopes, there has been a demand for a technique enabling observation using a scanning electron microscope (SEM) with high resolution.
However, a sample to be inspected with an inspection apparatus incorporating SEM capabilities is normally placed in a sample chamber whose inside pressure has been reduced by vacuum pumping. The sample placed in the sample chamber which, in turn, is placed in a reduced-pressure ambient in this way is irradiated with an electron beam (charged-particle beam). Secondary signals, such as secondary electrons or backscattered electrons, produced from the sample in response to the irradiation are detected.
In such inspection of a sample using SEM, the sample is exposed to a reduced-pressure ambient. Therefore, moisture evaporates from the sample, so that the cells die. This makes it impossible to observe reactions to stimuli.
Accordingly, when an inspection is performed under the condition where the sample contains moisture, it is necessary to prevent the sample from being exposed to the reduced-pressure ambient; otherwise, moisture would evaporate from the sample. One conceivable method of inspecting a sample using SEM without exposing the sample to a reduced-pressure ambient in this way consists of preparing a sample holder whose opening (aperture) has been sealed off by a film, placing the sample in the holder, and installing the holder in an SEM sample chamber that is placed in the reduced-pressure chamber.
The inside of the sample holder in which the sample is placed is not evacuated. The film that covers the opening formed in the sample holder can withstand the pressure difference between the reduced-pressure ambient inside the SEM sample chamber and the ambient (e.g., atmospheric-pressure ambient) of the inside of the sample holder that is not pumped down. Furthermore, the film permits an electron beam to pass therethrough (see JP-T-2004-515049).
When a sample is inspected, an electron beam is directed at the sample placed within the sample holder from outside the sample holder via the film on the sample holder placed in the SEM sample chamber that is in the reduced-pressure ambient. Backscattered electrons are produced from the irradiated sample. The backscattered electrons pass through the film on the sample holder and are detected by a backscattered electron detector mounted in the SEM sample chamber. Consequently, an SEM image is derived. However, with this technique, the sample is sealed in the closed space and so it has been impossible to give a stimulus to cells using a manipulator.
An example of a method of obtaining an SEM image by irradiating a sample with an electron beam via a film capable of withstanding the pressure difference between a vacuum and atmospheric pressure and detecting backscattered electrons emanating from the sample in this way is described also in Atmospheric Scanning Electron Microscopy, Green, Evan Drake Harriman, Ph. D., Stanford University, 1993 (especially Chapter 1: Introduction).
An example of a method of obtaining an image using a transmission electron microscope by placing a pair of films of the structure described above in an opposite relation to each other and placing the sample between the films is described in JP-A-47-24961 and JP-A-6-318445. Especially, JP-A-47-24961 makes a mention of a method of using such a pair of films. That is, an SEM image of a sample placed between the films is derived.
Morphological variations based on reactions of cells after a stimulus is given to the cells using a manipulator take place in microscopic regions within the cells. Therefore, the variations cannot be observed with an optical microscope. Hence, observation using SEM is essential. In order to observe the cells by SEM while maintaining the liquid, the sample (cells) is sealed in a sample holder. An electron beam is directed at the sample via a film formed on the sample holder, thus obtaining an image. However, with the aforementioned sample holder, the inside of the holder is a closed space. Consequently, it has been impossible to use a manipulator for giving a stimulus.